Pathologic alterations of chromatin structure promote tumorigenesis by affecting epigenomic and genomic stability and causing aberrant transcription. Both silent and active chromatin can spread over large genomic distances to create higher-order structures of extended islands and domains. We have found that the insulator protein CTCF binding sites define precise molecular boundaries at a number of large heterochromatin domains found in the human genome, including the HOXA locus, a cluster of homeo box genes known to be important in both development and disease. Epigenetic and genetic deregulations of the HOXA locus define a common oncogenic program in leukemia. We hypothesize that CTCF is required for establishment and maintenance of the proper chromatin structure and higher order organization at the HOXA locus. To examine CTCF function at the HOXA locus, we will employ a battery of molecular analyses, including reporter assays, BAC recombineering, chromatin immunoprecipitation, and chromosome conformation capture. Detailed analyses of the HOXA locus will enable us to understand biochemical and molecular mechanisms underlying the large-scale organization of chromatin and to decipher how alteration of CTCF function might lead to altered expression of HOXA genes which is frequently observed in cancer cells. In parallel, we will use biochemical and proteomic approaches to identify trans-acting factors that are differentially and uniquely associated with the CTCF binding site at the HOXA locus, and utilize short interfering RNA mediated ablation to study their function. Lastly, we will characterize the effect of cancer-associated mutations in the CTCF gene and ectopic expression of the competitive paralog BORIS at the HOXA locus and throughout the entire human genome. These proposed experiments represent an integrated effort to determine the function of CTCF in restricting heterochromatin and euchromatin, to uncover additional mechanisms that control genome expression, and to extend our understanding of the human genome and epigenome structure and function. This project will provide critical insights into mechanisms underlying cancer-promoting alterations of chromatin structures and will provide new drug targets responsible for the cancer epigenome.